CN115792032B

CN115792032B - A UPLC-MS/MS method for simultaneous determination of multiple tetrabromodiphenyl ether metabolites in aquatic products

Info

Publication number: CN115792032B
Application number: CN202211650217.8A
Authority: CN
Inventors: 耿倩倩; 谭志军; 郭萌萌; 崔正国; 邹亮; 曲梦
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2022-12-21
Filing date: 2022-12-21
Publication date: 2025-02-25
Anticipated expiration: 2042-12-21
Also published as: CN115792032A

Abstract

The invention discloses a UPLC-MS/MS method for simultaneously determining a plurality of tetrabromodiphenyl ether metabolites in aquatic products, and specifically relates to the technical field of aquatic product pollution detection, comprising the following steps: step 1, sample extraction, S1: accurately weighing 5.0±0.2g of a homogenized sample, and sequentially adding 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate; S2: vortexing the sample solution treated in S1 for 25-32s, placing it in a water bath for ultrasonic-assisted extraction for 9-13min, centrifuging at 4000rpm for 5min, and taking the supernatant into a new tube; S3: adding 4-6mL of the above-mentioned acetone-n-hexane to the new tube containing the supernatant in S2, repeating the extraction once, centrifuging at 4000rpm for 4-6min, and combining the supernatants. Aiming at the matrix characteristics of aquatic products, the present invention combines mixed solvent-ultrasound-assisted liquid-liquid extraction and solid phase extraction purification methods to establish a high-throughput UPLC-MS/MS detection method for various BDE-47 metabolites in aquatic products, which is rapid, sensitive and accurate.

Description

UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products

Technical Field

The invention relates to the technical field of aquatic product pollution detection, in particular to a UPLC-MS/MS method for simultaneously measuring various tetrabromodiphenyl ether metabolites in aquatic products.

Background

Persistent Organic Pollutants (POPs) have the characteristics of wide distribution, persistence, multiple toxicity and the like, and can be accumulated and amplified through a food chain to seriously harm human bodies and ecological environment. Polybrominated diphenyl ethers (PBDEs) are toxic halogenated flame retardants of great current interest and are widely detected in environmental media and organisms. Wherein 4 PBDEs and HBCD containing 4-7 bromine atoms have been listed in the POPs list. PBDEs can be accumulated and amplified through food chains, and PBDEs with different concentrations are detected in the bodies of a plurality of aquatic products such as fish, shellfish and the like.

2,2', 4' -Tetrabromodiphenyl ether (BDE-47) is one of PBDEs homologs with higher abundance in the environment, has wider distribution in natural environment, particularly marine environment, has bioaccumulation property and stronger neurotoxicity, genotoxicity, immunotoxicity and the like, and belongs to novel POPs. Toxicology studies have shown that both the methoxylated BDE-47 (MeO-BDE 47) and the hydroxylated BDE-47 (HO-BDE 47) derivatives of BDE-47 have a higher toxic effect than the parent compound BDE-47 itself, consuming aquatic products contaminated with BDE-47 and its metabolites or causing serious health problems.

At present, no relevant standard is found in detection methods for determining BDE-47 hydroxylation and methoxy derivatives in aquatic products. The method for measuring the hydroxylation and methoxy BDE-47 comprises a gas chromatography-mass spectrometry (GC-MS) method and a liquid chromatography tandem mass spectrometry (HPLC-MS) method, and is mainly characterized by single metabolite index measurement, and has the advantages of complicated steps, time-consuming operation and longer detection period, such as the GC-MS method needs to be added with a silane reagent for derivatization, has more interference factors, and cannot meet the actual requirements of rapid and accurate multi-sample multi-index measurement. The aquatic products are rich in oil, protein and other interference impurities, and the complex substrate interferes with the detection and analysis of the instrument.

Disclosure of Invention

This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.

The present invention has been made in view of the above-mentioned problems associated with the conventional UPLC-MS/MS method for simultaneously determining a plurality of tetrabromodiphenyl ether metabolites in an aquatic product.

Therefore, the invention aims to provide a UPLC-MS/MS method for simultaneously measuring various tetrabromodiphenyl ether metabolites in aquatic products, which aims to solve the problems that the measurement is mainly carried out by using metabolite indexes, the steps are complicated, the operation is time-consuming, the detection period is long, in addition, if silane reagent is added for derivatization, the interference factor is large, the actual requirement of rapid and accurate measurement of multiple samples and multiple indexes cannot be met, the aquatic products are rich in oil, protein and other interference impurities, and the complex substrate interferes the detection and analysis of an instrument.

In order to solve the technical problems, the invention provides a UPLC-MS/MS method for simultaneously measuring a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, which comprises the following steps:

step one, extracting a sample

S1, accurately weighing 5.0+/-0.2 g of homogenized sample, and sequentially adding 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride and 0.8-1.1g of anhydrous sodium sulfate;

s2, carrying out vortex oscillation on the sample solution treated in the step S1 for 25-32S, then placing the sample solution in a water bath for ultrasonic-assisted extraction for 9-13min, centrifuging at 4000rpm for 5min, and taking supernatant to a new tube;

s3, adding 4-6mL of acetone-n-hexane into the new pipe filled with the supernatant in the step S2, repeatedly extracting once, centrifuging at 4000rpm for 4-6min, and combining the supernatants.

Step two, purifying the sample solution

5ML of the combined supernatant was loaded on an activated CAPTIVA EMR-Lipid (6 mL,600 mg) solid phase extraction cartridge, the flow rate was maintained at about 2mL/min, the effluent was collected, nitrogen blown to near dryness at 38-42℃and acetonitrile was added to a constant volume of 0.7-1.3mL.

Step three, testing the sample solution by adopting liquid chromatography-tandem mass spectrometry

Wherein, the liquid chromatography conditions are as follows:

The chromatographic column is Waters ACQUITY UPLC BEH C chromatographic columns, the column temperature is 33-36 ℃, the sample injection amount is 5 mu L, the mobile phase A is 0.1% formic acid aqueous solution (containing 2mmol of ammonium acetate), and the mobile phase B is acetonitrile. Gradient elution procedure was as follows, 0-1min, a: b=70:30, 1-2min, a: b=50:50, 2-5min, a: b=5:95, 5-10min, a: b=5:95, 10-11min, a: b=70:30.

Wherein, the mass spectrum condition is:

The temperature of the drying gas is 280-310 ℃, the flow rate of the drying gas is 9-12L/min, the atomization gas pressure is 40psi, the capillary voltage is 3500V, the sheath flow temperature is 350 ℃, and the sheath flow velocity is 12L/min.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, the invention is characterized in that in S1, 10mL of acetone-n-hexane is added, and v/v=1:1 of the acetone and the n-hexane.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, the invention is characterized in that in S1, the weight of added sodium chloride is 1g, and the added anhydrous sodium sulfate is 1g.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, the invention comprises the steps of in S2, carrying out vortex oscillation on a sample solution for 30 seconds, and then placing the sample solution in a water bath for ultrasonic-assisted extraction for 10min.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, the invention is characterized in that 5mL of acetone-n-hexane is added for repeated extraction in S3, and centrifugation is carried out at 4000rpm for 5min.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring various tetrabromodiphenyl ether metabolites in aquatic products, the invention comprises the steps of collecting effluent liquid in the second step, blowing nitrogen to near dryness at 40 ℃, and adding acetonitrile to fix the volume to 1mL.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, the invention comprises the step three, wherein the temperature of a chromatographic column under the liquid chromatography condition is 35 ℃.

As a preferable scheme of the UPLC-MS/MS method for simultaneously measuring various tetrabromodiphenyl ether metabolites in aquatic products, the invention comprises the following steps of drying gas temperature of 300 ℃ and drying gas flow of 10L/min under the mass spectrum condition in the third step.

In summary, the present invention includes at least one of the following beneficial effects:

aiming at the characteristics of aquatic product substrates, the invention combines the mixed solvent-ultrasonic auxiliary liquid-liquid extraction and solid phase extraction purification means to establish a high-throughput UPLC-MS/MS detection method for various BDE-47 metabolites in the aquatic products, and has the characteristics of rapidness, sensitivity and accuracy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to these drawings for a person having ordinary skill in the art.

FIG. 1 is a MRM profile of a measured metabolite of the invention;

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention discloses a UPLC-MSMS method for simultaneously measuring various tetrabromodiphenyl ether metabolites in aquatic products.

Example 1

A UPLC-MS/MS method for simultaneously determining a plurality of tetrabromodiphenyl ether metabolites in an aquatic product, comprising the steps of:

step one, extracting a sample

Step two, purifying the sample solution

Wherein, the liquid chromatography conditions are as follows:

The chromatographic column is Waters ACQUITY UPLC BEHC chromatographic columns, the column temperature is 33-36 ℃, the sample injection amount is 5 mu L, the mobile phase A is 0.1% formic acid aqueous solution (containing 2mmol of ammonium acetate), and the mobile phase B is acetonitrile. Gradient elution procedure was as follows, 0-1min, a: b=70:30, 1-2min, a: b=50:50, 2-5min, a: b=5:95, 5-10min, a: b=5:95, 10-11min, a: b=70:30.

Wherein, the mass spectrum condition is:

The specific values in the above steps are as follows, in S1, the added acetone-n-hexane is 8mL, the v/v=1:1 of acetone and n-hexane, the added sodium chloride is 0.7g, the added anhydrous sodium sulfate is 0.8g, in S2, the sample solution is subjected to vortex oscillation for 25S and then is placed in a water bath for ultrasonic auxiliary extraction for 9min, in S3, the added acetone-n-hexane is 4mL for repeated extraction once, the centrifugation is carried out at 4000rpm for 4min, the effluent liquid collected in the second step is blown to near dryness at 38 ℃, the acetonitrile is added to constant volume to 0.7mL, the chromatographic column temperature of the liquid chromatographic conditions in the third step is 33 ℃, the dry gas temperature of the mass spectrum conditions in the third step is 280 ℃, and the dry gas flow is 9L/min.

Example two

step one, extracting a sample

Step two, purifying the sample solution

Wherein, the liquid chromatography conditions are as follows:

Wherein, the mass spectrum condition is:

The specific numerical values in the steps are as follows, in S1, 10mL of acetone-n-hexane is added, v/v=1:1 of acetone and n-hexane, 1g of sodium chloride is added, 1g of anhydrous sodium sulfate is added, in S2, the sample solution is subjected to vortex oscillation for 30 seconds and then is placed in a water bath for ultrasonic auxiliary extraction for 10min, in S3, 5mL of acetone-n-hexane is added for repeated extraction once, centrifugation is carried out at 4000rpm for 5min, effluent liquid is collected in the second step, nitrogen is blown to near dryness at 40 ℃, acetonitrile is added to reach 1mL, the column temperature of a liquid chromatography condition in the third step is 35 ℃, the drying gas temperature of a mass spectrum condition in the third step is 300 ℃, and the drying gas flow rate is 10L/min.

Example III

step one, extracting a sample

Step two, purifying the sample solution

Wherein, the liquid chromatography conditions are as follows:

Wherein, the mass spectrum condition is:

The specific values in the steps are as follows, in S1, 11mL of acetone-n-hexane is added, v/v=1:1 of acetone and n-hexane, the weight of added sodium chloride is 1.3g, the added anhydrous sodium sulfate is 1.1g, in S2, the sample solution is subjected to vortex oscillation for 32S and then is placed in a water bath for ultrasonic auxiliary extraction for 13min, in S3, 6mL of acetone-n-hexane is added for repeated extraction once, centrifugation is carried out at 4000rpm for 6min, the effluent liquid is collected in the step II, nitrogen is blown to near dryness at 42 ℃, acetonitrile is added to fix the volume to 1.3mL, the column temperature of the liquid chromatographic condition in the step III is 36 ℃, the dry gas temperature of the mass spectrum condition in the step III is 310 ℃, and the dry gas flow is 12L/min.

By analyzing the differences between the values in the different steps of the three embodiments, the value in the second embodiment is the optimal value, and the quantitative and qualitative ion pairs in the second embodiment are shown in the following table.

In addition, the linear correlation coefficient (R ²) of the 6 metabolites in the range of 0.2-20ng/mL in the second embodiment is 0.996-0.999, and the linear correlation is good. The detection limit is 0.1-0.3 mug/kg, the quantitative limit is 0.3-0.9 mug/kg, and the method has higher sensitivity and is concretely shown in the following table.

In the description of the present application, it should be noted that, unless otherwise specified and limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be mechanical or electrical, or may be a direct connection between two elements, where "up," "down," "left," "right," etc. are merely used to indicate relative positional relationships, and when the absolute position of an object to be described changes, the relative positional relationships may change;

In the drawings of the disclosed embodiments, only the structures related to the embodiments of the present disclosure are referred to, and other structures can refer to the common design, so that the same embodiment and different embodiments of the present disclosure can be combined with each other without conflict;

Finally, the foregoing description of the preferred embodiment of the invention is provided for the purpose of illustration only, and is not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

1. A UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products, characterized in that it comprises the following steps:

Step 1: Sample extraction

S1: Accurately weigh 5.0±0.2g of the homogenized sample, and add 8-11mL of acetone-n-hexane, 0.7-1.3g of sodium chloride, and 0.8-1.1g of anhydrous sodium sulfate in sequence;

S2: The sample solution treated in S1 was vortexed for 25-32 seconds and then placed in a water bath for ultrasound-assisted extraction for 9-13 minutes. The solution was centrifuged at 4000 rpm for 5 minutes and the supernatant was transferred to a new tube.

S3: Add 4-6 mL of the above acetone-n-hexane to the new tube containing the supernatant in S2 and repeat the extraction once, centrifuge at 4000 rpm for 4-6 min, and combine the supernatants;

Step 2: Purification of sample solution

Take 5 mL of the combined supernatant and load it onto the activated Captiva EMR-Lipid, 6 mL, 600 mg solid phase extraction column. Keep the flow rate at about 2 mL/min, collect the effluent, blow it to near dryness with nitrogen at 38°C-42°C, and add acetonitrile to make up to 0.7-1.3 mL.

Step 3: Test the sample solution using liquid chromatography-tandem mass spectrometry

The liquid chromatography conditions are:

The chromatographic column was a Waters ACQUITY UPLC BEH C18 column, the column temperature was 33°C-36°C, the injection volume was 5 μL, the mobile phase A was 0.1% formic acid aqueous solution containing 2 mmol ammonium acetate, and the mobile phase B was acetonitrile; the gradient elution program was as follows: 0-1 min, A:B=70:30; 1-2 min, A:B=50:50; 2-5 min, A:B=5:95; 5-10 min, A:B=5:95; 10-11 min, A:B=70:30;

The mass spectrometry conditions are:

Drying gas temperature: 280℃-310℃; Drying gas flow rate: 9-12L/min; Nebulizing gas pressure: 40psi; Capillary voltage: 3500V; Sheath gas temperature: 350℃; Sheath gas flow rate: 12L/min;

Among them, the six metabolites are 3-HO-BDE47, 5-HO-BDE47, 6-HO-BDE47, 3-McO-BDE47, 5-MeO-BDE47 and 6-MeO-BDE47.

2. The UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that in S1, 10 mL of acetone-n-hexane is added, and the v/v ratio of acetone to n-hexane is 1:1.

3. The UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that in S1, the weight of the added sodium chloride is 1 g, and the added anhydrous sodium sulfate is 1 g.

4. A UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that in S2, the sample solution is vortexed for 30 seconds and then placed in a water bath for ultrasound-assisted extraction for 10 minutes.

5. The UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that in S3, 5 mL of the acetone-n-hexane is added and the extraction is repeated once, and centrifuged at 4000 rpm for 5 min.

6. A UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that the collected effluent in step 2 is blown to near dryness with nitrogen at 40°C, and acetonitrile is added to make the volume to 1 mL.

7. The UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that the column temperature of the chromatographic column in the liquid chromatography condition in step 3 is 35°C.

8. The UPLC-MS/MS method for simultaneously determining multiple tetrabromodiphenyl ether metabolites in aquatic products according to claim 1, characterized in that the drying gas temperature of the mass spectrometry conditions in step 3 is 300°C; and the drying gas flow rate is 10 L/min.